Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36 |
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Authors: | Daniel F. Visser Fritha Hennessy Konanani Rashamuse Maureen E. Louw Dean Brady |
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Affiliation: | (1) Enzyme Technologies Group, CSIR Biosciences, Private Bag X2, Modderfontein, Johannesburg, 1645, South Africa;(2) Microbial Expression Systems, CSIR Biosciences, PO Box 395, Pretoria, 0001, South Africa; |
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Abstract: | A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V max of 2.03 × 10−9 s −1 and a K m of 206 μM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70°C and the enzyme had a half life at 60°C of 20.8 h. |
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